Preparation and evaluation of the immune efficacy of an inactivated fowl adenovirus 8a serotype oil emulsion vaccine

In recent years, fowl adenovirus (FAdV) transmission has significantly increased worldwide, leading to substantial economic losses in the poultry industry. The virus causes hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH). The prevalent FAdV strains in China are FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11. Vaccines for FAdV-4 and FAdV-8b, which prevent HHS and IBH, are available commercially, but no vaccine exists for FAdV-8a. To address this issue, we developed a vaccine using an oil emulsion to inactivate the FAdV-8a serotype. Additionally, we built a fluorescence quantitative PCR for the detection of the virus. The lowest concentration detected was 4.11 × 101 copies/μL. The study's results illustrated that the FAdV-8a oil emulsion vaccine effectively produced significant antibodies and offered ample protection for poultry. This vaccine can potentially limit the transmission of IBH resulting from FAdV-8a in China.

FAdV is responsible for widespread sickness and significant economic losses [5], and it can be transmitted vertically [6] through the yolk sac, chick embryo chorioallantoic membrane, and allantoic cavity, as well as horizontally via contaminated feed, water, or contact [7].Vertical transmission is considered the primary spread method, and the virus replicates primarily in chickens' digestive and respiratory tracts [8].The disease can be prevalent year-round, with a peak incidence during the summer and rainy season.IBH outbreaks have increased in the past two decades, with the virus responsible for its global spread [9].IBH primarily affects broiler chickens under 7 weeks of age [10], while layer hen infections have been reported occasionally.Because IBH-infected chickens often have concurrent disorders, some of the characteristics of field outbreaks are difficult to replicate in the laboratory.Among the 12 serotypes of FAdV, the most common serotypes of IBH in chickens are FAdV D and FAdV E [11].FAdV-4, FAdV-8a, and FAdV-8b are the predominant serotypes in China.IBH is characterized by a large, swollen, friable liver with a marbled subepithelial reticulum of fine linear and stellate hemorrhages [2]; pale, bloated, mottled kidneys with a hemorrhagic renal cortex; and in some instances, an inflated, hemorrhagic spleen [12].Inclusion bodies are present, and hepatocytes develop steatosis [13].
Vaccination is presently the primary technique for disease prevention and control [14], although cross-protection between FAdV serotypes is limited [15], and vaccine development is lacking.Inactivated vaccines are cheaper to produce, have a higher safety profile, and are easier to store and transport than other vaccines [16].Inactivated vaccines have been used on a larger scale against FadV than other types of vaccines [17].
Inactivated vaccines are a type of vaccine that uses chemical or physical methods to inactivate the pathogen during preparation.Compared to other types of vaccines, inactivated vaccines are less costly to produce and have the advantage of being safer and easier to store and transport.Viruses are killed during the preparation of inactivated vaccines and lose their biological activity.These viruses retain their original structure and antigenicity but can no longer replicate or cause infection.Inactivated vaccines are, therefore, effective in inducing the body's immune system to produce specific antibodies, thereby preventing the onset and spread of disease.Inactivated vaccines are more accessible to apply than other types of vaccines, and they do not require special storage conditions or cold chain transport, making them more easily distributed around the globe, especially in areas where medical resources are scarce, or infrastructure is inadequate [18].
A highly effective inactivated oil emulsion vaccine has been successfully developed in the present study using FAdV-8a.The virus was isolated from the livers of FAdV-infected chickens.Test results showed that the vaccine elicited a robust immune response in SPF chickens after immunization.The vaccine also demonstrated excellent immunoprotective efficacy, ultimately inhibiting the cloacal shedding phenomenon, and showed no safety concerns.This vaccine is a promising candidate strain for developing FAdV-8a vaccines with broad market application.

Virus and cell line
The FAdV-8a strain CY21 used in this study was taken from a broiler farm in Shandong Province.The farm was experiencing a disease called inclusion body hepatitis (IBH), and the Poultry Disease Laboratory obtained the isolate at the Shandong Academy of Agricultural Sciences.The chicken Leghorn male hepatoma (LMH) cell line (ATCC #CRL-2117) was used for cell culture, and it was grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (PAN, Germany) and 1% penicillin-streptomycin.The cells were maintained at 37 • C in a humidified environment with 5% CO2.

Virus passage and titration
LMH cell lines were used to grow the virus in 25 cm2 cell culture flasks.When the cells reached about 80% confluence, the culture medium was removed, and the cells were washed with phosphate-buffered saline (PBS).Then, 200 μL of a 100-fold diluted virus solution was added and incubated for an hour.After that, 5 mL of maintenance medium containing 1% fetal bovine serum (FBS) was added, and the cytopathic effect was monitored daily.The CY21 strain was passaged continuously for 15 generations in LMH cells.Virulence values were measured for the 3rd, 6th, 9th, 12th, and 15th generations to create a virulence change curve.Also, generations with high virulence were inoculated into LMH cells, and samples were taken at 6, 12, 18, 24, 36, 48, 60, 72, 84, and 96 h after injection to produce a growth curve.

Preparation of oil-adjuvant inactivated FAdV-8a vaccine
The antigen was inactivated using a final concentration of 0.2% formaldehyde.It was then added to the culture medium obtained from LMH cells infected with FAdV-8a.The inactivated antigen solution was mixed with white oil (HengFa, Guangzhou, China) at a ratio of 1:3.This mixture was then centrifuged at 6000 rpm for 10 min in a high-speed centrifuge.

Animal experiment
To evaluate the effectiveness of the inactivated FAdV-8a vaccine, 32 specific pathogen-free (SPF) chickens were used in the study.The chickens were 21 days old and kept in separate positive-pressure isolators.They were randomly divided into four groups, each containing eight chickens.Group A was given 0.3 mL of the vaccine containing 10 6.5 TCID 50 /0.1 mL via intramuscular injection.Group B and Group C were given 0.3 mL of the vaccine containing 10 5.5 TCID 50 /0.1 mL and 10 4.5 TCID 50 /0.1 mL, respectively.Group D was given 0.3 mL of a mixture of DMEM and white oil emulsion at a 1:3 ratio and was used as the unvaccinated control.
On Day 21, after infection, blood samples were taken from all the chickens and tested with the BioChek FAdV-I antibody ELISA kit from BioChek (China).The viral challenge involved the administration of FAdV-8a-CY21 (10 6 TCID 50 /bird) intramuscularly at a dose of 0.3 mL per chicken.
We collected cloacal swabs from five chickens in each group during the study at 3, 6, 9, 12, and 15 days after infection.The samples were placed in 2 mL centrifuge tubes, and 1 mL of saline was added to each tube.After shaking the tubes, they were subjected to two freeze-thaw cycles and centrifuged at 8000 rpm for 15 min.We collected the supernatant and used fluorescence quantitative PCR for shedding detection.
Three chickens from each group were examined 6 days post-infection (dpi) to observe organ lesions.The heart, liver, spleen, lung, kidney, brain, bursa, and duodenum were collected, weighed, and homogenized in a saline solution with a ratio of 1:10.The homogenate was subjected to two freeze-thaw cycles, followed by centrifugation at 8000 rpm for 15 min.The supernatant was collected for detection using fluorescence quantitative PCR.

The viral load and growth curve of CY21
According to the Reed-Muench methodology, the viral TCID 50 was calculated.CY21's titers remained at a TCID 50 of 10 8.0 /mL after the 9th generation (Fig. 1A), and it reached its highest virulence of TCID 50 10 8.0 /mL 72 h after incubation (Fig. 1B).

Antibody responses of vaccinated chickens
The results of the study are presented in Fig. 2. It shows that all chickens in Group A had high levels of antibodies compared to the control group (p < 0.0001).In contrast, only half of the chickens in Group B had low levels of antibodies compared to the control group (p < 0.05).Out of all the chickens in Group C, only two showed positive results, while all chickens in Group D had negative results.

Clinical symptoms and gross pathology
During the experiment, the chickens in Groups A and B didn't show any clinical signs, while those in Groups C and D showed depression and decreased feeding at 3 days post-infection (dpi).However, none of the chickens died during the experiment.At 6 dpi, three chickens from each group underwent autopsy.Upon visual inspection, Groups A (Fig. 3A) and Groups B (Fig. 3B) showed no obvious lesion symptoms.On the other hand, the livers of chickens in Group C appeared swollen, turned pale yellow, and had red bleeding spots visible on the surface (Fig. 3C).The livers of chickens in Group D were also swollen with bluntly rounded edges, had bleeding spots on the surface, and became brittle and friable (Fig. 3D).

Standard curves
After optimization, we established the reaction system and conditions for the fluorescence PCR assay.The reaction system was composed of 10 μL of TB Green Premix Ex, with a final concentration of 0.1 μM for both upstream and downstream primers, and 2 μL of DNA template, adjusted to a final volume of 20 μL with water.
The reaction conditions consisted of a predenaturation step of 30 s at 95 We generated standard curves using plasmid DNA with concentrations ranging from 4 × 10 6 to 4 × 10 1 copies/μL (Fig. 4A).The linear regression equation for the standard curve had a correlation coefficient of 0.998 and an amplification efficiency of 103.9% (Fig. 4B).

Specificity
The assay showed high specificity, with only FAdV-8a-CY21 yielding positive results, while all other samples and the blank control were negative or had CT values exceeding 36.59, as shown in Fig. 5.

Sensibility
Recombinant plasmids with a concentration ranging from 4.11 × 10 1 to 4.11 × 10 5 copies/μL at 10-fold dilution were used as templates for the assay.The fluorescence PCR method detected a minimum concentration of 4.11 × 10 1 copies/μL, as shown in Fig. 6.

Fig. 2.
Antibody responses of vaccinated chickens (n = 8 birds for each construct).Group A was immunized with 10 6.5 TCID 50 /0.1 mL.Group B was immunized with 10 5.5 TCID 50 /0.1 mL.Group C was immunized with 10 4.5 /0.1 mL.Group D was the negative control.

Detection of viral load in tissues
The results of the study are presented in Fig. 7. Viral DNA was not found in the kidney or brain.Group A showed no viral DNA in any organs, while Group B had trace amounts of viral DNA only in the bursa.Both Groups C and D exhibited viral DNA detection in the heart, liver, spleen, lung, duodenum, and bursa.The duodenum showed the highest peak with a viral copy number of 4 × 104.3 copies/ μL.There was no viral DNA detected in the brain or kidney.

Detection of shedding
The results indicate that reducing the antigen titer to less than 10 4.5 TCID 50 /0.1 mL (100-fold dilution group) does not prevent the shedding of the challenge test chickens.Similarly, an increase to 10 5.5 TCID 50 /0.1 mL (10-fold dilution group) only partially inhibits shedding by 40%.However, when the antigen titer reaches 10 6.5 TCID 50 /0.1 mL (in the primary fluid it completely suppresses the shedding of the challenge test chickens (Table 1).These findings suggest that the inactive CY21 vaccine is highly immunogenic, as it not only stimulates high levels of antibodies but also effectively inhibits shedding, demonstrating its robustness.
A study has shown that FAdV-8a and FAdV-11 offer partial cross-protection, while there is no cross-protection between FAdV-8b and FAdV-11 [37].Therefore, developing a FAdV-8a vaccine can partially protect against FAdV-8b and FAdV-11 infections and has the potential for promising vaccine candidate vaccines.
In this investigation, we prepared a vaccine utilizing the virulent FAdV-8a CY21 strain, which was clinically isolated and inactivated via an oil emulsion.The serum obtained on Day 21 after a single immunization displayed a high immune antibody level.These results are consistent with the findings of P.A. Steer-Cope et al. [37], which demonstrated that the inactivated FAdV-8a vaccine elicited serum antibodies that neutralized FAdV-8a with titers ranging from 600 to 20,000 after immunization.Moreover, the cloacal shedding assay revealed that when the antigen titer reached 10 4.5 TCID 50 /0.1 mL, it could not inhibit the shedding of challenge test chickens.These observations highlight that when the virulence of the immunogen got a certain threshold, the CY21 inactivated vaccine elicited a  Viral titers in samples (heart, liver, spleen, lung, kidney, brain, duodenum, and bursa) from FAdV-8a-infected chickens(n = 3 birds for each construct).Group A was immunized with 10 6.5 TCID 50 /0.1 mL.Group B was immunized with 10 5.5 TCID 50 /0.1 mL.Group C was immunized with 10 4.5 TCID 50 /0.1 mL.Group D was an unvaccinated control.

Table 1
Virus detection in each group after the challenge.high level of antibody response, thereby effectively inhibiting the replication and transmission of the virus.Thus, the vaccine exhibited good immunogenicity and immunoprotective effects, thereby holding potential for developing FAdV-8a vaccine candidates.In addition, related studies suggest that the protective effects of the FAdV-8a virus may extend to serotype D [37].Further research is needed to determine whether the inactivated vaccine of the CY21 strain prepared in this study has protective effects against other serotypes.The focus of this study is the evaluation of antibody levels and vaccine protective efficacy after a single vaccine immunization using an inactivated FAdV-8a oil emulsion vaccine, which is the first of its kind in China.It is noteworthy that certain aspects of this study merit further exploration, including the applicability of the vaccine to other serotypes of the virus and its long-term sustainability in practical application.These areas require thorough investigation through additional studies to enhance this vaccine's theoretical foundation and efficacy.

Fig. 1 .
Fig. 1.Growth curve of FAdV-8a and the change in the toxic value curve in LMH cells.(A) Changes in FAdV-8a virulence in successive passages of LMH cells.(B) Changes in FadV-8a virulence over time.
• C, followed by denaturation at 95 • C for 10 s, annealing at 58 • C for 15 s, extension at 72 • C for 15 s, and 40 cycles.During each extension cycle, fluorescence signals were collected at 72 • C.